以大鼠腦血管內皮細胞 (ARBEC) 探討導致巴金森氏症之相關因子通透血腦屏障之機轉及其影響

Abstract

To date, compounds including MPTP/MPP+, paraquat and rotenone have been reported to be related to Parkinson’s disease (PD). On the other hand, nicotine may have neuroprotective effect on PD. Previously, using adult rat brain endothelial cell (ARBEC), we showed that nicotine competitively inhibited cellular uptake of MPP+. In this regard, the current study further examines the interaction between MPTP/MPP+ and several compounds, including nicotine, organic cations and MPTP/MPP+ analogues in ARBEC. In addition, since paraquat was reported to penetrate blood-brain barrier via large neutral amino acid transport 1 (LAT1), in the second part of this study, we used cell culture (ARBEC) model to further examine the interaction between LAT1 and paraquat. In the study of MPTP/MPP+, firstly, the results showed that cellular uptake of MPTP can be mediated by a carrier-mediated system, with Km and Vmax value of 35.7 ± 0.9 μM and 1228.4 ± 106.5 pmole/mg protein-30 sec, respectively. Secondly, the carrier system transported MPTP/MPP+ and nicotine was sensitive to decynium22, a selective organic cation transporter (OCT) inhibitor. RT-PCR showed that OCT1 mRNA was detectable in ARBEC. Thirdly, not only nicotine but also amantadine and selegiline can inhibit the cellular uptake of MPTP and MPP+, whereas caffeine and levodopa did not have any significant effect. Nicotine, amantadine and selegiline competitively inhibited cellular uptake of MPTP with inhibition constants (Ki) of 329 μM, 178 μM and 246 μM respectively. In addition, MPTP/MPP+ analogues such as tetrahydroisoquinoline (TIQ) and N-methyl-salsolinol showed significant inhibition on cellular uptake of MPTP, MPP+ and nicotine. These results indicated that nicotine, amantadine and selegiline may inhibit MPTP-like neurotoxins across the blood-brain barrier. Nonetheless, due to structural similarity, BBB transfer characteristics and structure-transport relationship for MPTP/MPP+ analogues need to be further elucidated. In the study of paraquat, different from the reports in literature, our result showed that LAT1 did not recognize and transport paraquat into the brain endothelial cell. Instead, paraquat seems to enter ARBEC by passive diffusion. On the other hand, neonatal rat astrocytes (NRA) can uptake paraquat via a carrier-mediated process. Since NRA was considered a secondary barrier in the BBB constitute, further study is required to investigate whether NRA plays a role in the transport of paraquat into the CNS. In conclusion, our results showed that nicotine could competitively inhibit cellular uptake of MPTP. It might partly explain the neuroprotective effect of nicotine in PD. In addition to nicotine, amantadine and selegiline may exhibit the same neuroprotective effect. On the other hand, our results showed that paraquat is not transported by LAT1. The transport mechanisms of paraquat through BBB should be further varified.